Field of the Invention
The present invention relates to diagnostic reagents for immunological tests. In particular, the invention relates to an improvement in or relating to a particulate carrier and diagnostic reagents for immunological tests for detecting or measuring a component in human or animal body fluids or for labeling cells, and especially relates to immunoparticles prepared by immobilizing immunochemicals on a particulate carrier.
In immunologically detecting or quantitatively analyzing either an antigen or antibody using the reaction between the antigen and antibody, an important method in immunological tests in clinical laboratories is the method of immobilizing a substance which reacts with a second substance to be detected, on a particulate carrier and carrying out a high sensitivity measurement utilizing the phenomenon of agglutination of said immobilized substance-carrier combination particles in the presence of the substance to be detected. Another method which has also been broadly used in clinical laboratory tests is the method of immobilizing a substance to be detected on a particulate carrier, utilizing the fact that agglutination, of particles immobilizing the substance to be detected due to the presence of an antigen or antibody specifically reacting with the substance to be detected, is inhibited by the presence of the substance to be measured in the body fluids, thereby detecting or quantitatively analyzing the substance to be detected. The latter method is often called the agglutination inhibition method. In addition, cell labeling has been frequently used for immunological testing. Cell labeling is the method of immobilizing a substance, which selectively binds to specific cells, on a particulate carrier and labeling the cells by determining whether or not the particles bind to the cells.
Such immobilized substance-particulate carrier combinations are referred to as "immunoparticles".
As a particulate carrier as a part of a diagnostic reagent for immunological tests using such immunoparticles for agglutination reaction, substances used include red corpuscles of mammals, including man and birds, particles of an inorganic substance such as kaolin and carbon, and latex of organic high polymers such as natural rubber latex and polystyrene latex. Of these, red corpuscles can immobilize many kinds of antigens and antibodies and the applicable range thereof is the broadest. However, red corpuscles have disadvantages in that there are differences in quality depending upon the individual animals from which it is selected. Red corpuscles are also difficult to store and they may be non-specifically agglutinated by human serum.
The most widely used non-organism originating carrier particles are polystyrene particles. Polystyrene is stable and because it is a synthetic polymer, the quality can be controlled. Because polystyrene is hydrophobic and has properties of adsorbing various proteins, immobilization of an antigen or antibody on polystyrene is usually carried out by physical adsorption. When an antigen or antibody is immobilized by physical adsorption, an euilibrium may occur between the immobilized antigen (or antibody) and a free antigen (or antibody) and result in a competitive reaction which takes place between the antigen (or antibody) immobilized on particles and the free antigen (or antibody) toward a corresponding antibody (or antigen) which is an objective substance of the measurement. This competitive reaction works to inhibit agglutination. As a result, insufficient sensitivity and stability occurs in many instances. Moreover, when the protein component in the body fluids of an object of detection is adsorbed by the polystyrene, agglutination takes place even when the objective reaction between the antigen and the antibody does not take place and a biological false-positive reading results. Also, substances incapable of being physically adsorbed to polystyrene cannot be immobilized by this method. Because of these disadvantages, polystyrene particles are used practically only within a limited range.
In order to overcome these difficulties, it has been proposed to use other reagents bonding an antigen or antibody to a carrier by covalent bonding. Reagents which bond human chorionic gonadtropin to a styrene--methacrylic acid copolymer latex using carbodiimide (DT No. 2,649,218), reagents consisting of particles having diameters of 0.01-0.9 microns bonding human chorionic gonadtropin, human serum albumin or aggregated .gamma.-globulin to various latices via amide bond such as carboxylated styrene--butadiene copolymer, carboxylated polystyrene, carboxylated polystyrene having an amino group, acrylic acid polymer, acrylonitrile polymer, methacrylic acid polymer, acrylonitrile-butadiene-styrene terpolymer, polyvinyl acetate acrylate, polyvinyl pyridine and vinyl chloride--acrylate copolymer (Japanese Patent Application Publication No. 12966/1978) and reagents obtained by copolymerizing methyl methacrylate, 2-hydroxyethyl methacrylate and methacrylic acid and bonding treponema antigen to a latex of the copolymer containing hydroxyl group and carboxyl group prepared by the copolymerization by cyanogen bromide or carbodiimide method ("The Japanese Journal of Clinical Pathology", 27, Supplementary Edition, page 422 (1978), have been proposed.
Where many of the aforementioned carriers contain a large amount of hydrophobic portions in the main component of the polymer, for example, the styrene-methacrylic acid copolymer, they tend to adsorb protein, especially with blood plasma and serum where high concentrations of protein occur. When protein is adsorbed onto a carrier from the test body fluids, it may interfere with the objective reaction of the antigen and the antibody, causing a reduction in the selectivity or sensitivity of the agglutination reaction.
Some of the aforementioned carriers, for example, the acrylic acid polymer, are electrolytes. It is generally known that the existence of an electrolyte in a large amount weakens the binding of the antigen to the antibody. Further, an acrylic acid polymer and a methacrylic acid polymer ionizingly bond to protein high in isoelectric point pH. As mentioned above, it is disadvantageous to have protein other than the constitutional element of the objective immunological reaction non-selectively attached to the carrier. Accordingly, it is disadvantageous to use a polymer consisting mainly of an electrolyte as the carrier of a reagent for an agglutination reaction.